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 Detalle_Publicacion

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

Abstract: Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

 Fuente: J Vis Exp . 2020 Dec 5;(166).

Editorial: MYJoVE Corporation

 Año de publicación: 2020

Nº de páginas: 15

Tipo de publicación: Artículo de Revista

 DOI: 10.3791/59639

ISSN: 1940-087X

Autores/as

LI, HEGANG

QIN, HUAIYUAN

ZHANG, NING

ZHAO, JINSHAN

XIN, JINGJING

LIU, HUAWEI